Human respiratory syncytial virus (hRSV) is the leading cause of viral pneumonia, bronchiolitis, respiratory failure, mechanical ventilation, and viral death in infants in the USA and worldwide. There are no effective vaccines for hRSV disease. In order to identify new targets for drug screening, it is imperative that we gain more structural information regarding critical RNA and protein complexes in the virus life cycle, specifically during assembly of the infectious virion. Here we will investigate: 1) the structural and functional implications of cellular and viral membrane structure and composition for ordering hRSV assembly and scission of the virus from the cell membrane; 2) the in situ structures of complexes formed between F, G, M, M2-1, and the genomic RNA during hRSV assembly; and 3) develop labeling strategies specifically for cryo-EM/cryo-ET technologies to address the ultrastructural analyses of hRSV and many other viruses. To answer these questions, we are using the A2 strain of hRSV to infect permissive, model human-derived cells for correlative fluorescence microscopy and cryo-electron microscopy experiments. The three areas to be investigated are: 1. Determine the optimal strategy for labeling hRSV viral complexes assembling into viral particles in live cells. In this aim, we will develop, test, and use several strategies to label viral proteins in live cells in order to study virus assembly through correlatie fluorescence light microscopy (FLM) and cryo-electron tomography (cryo-ET) approaches. Alternative labeling strategies will include metallothionein - fluorescent protein conjugates, SNAP, and CLIP tags along with PEG-nanogold-benzylguanine or cytosine conjugates delivered to live cells. Efficient incorporation of the labeled protein into viral structures and vral titers similar to wild-type infections will be criterions for success. 2. Define the coordinated roe of the membrane and viral glycoproteins for fostering virus assembly and budding events. Experiments will determine if F and G reside within specific plasma membrane microdomains and if this facilitates recruitment of M, M2-1, and RNP complexes. Experiments will employ multiple cell types to examine and define any potential relationships, differences, or commonalities between cell type and virus replication. 3. Determine the structure of complexes formed between M, M2-1 and the ribonucleoprotein (RNP) complex during hRSV assembly. Experiments will determine the structures of the complexes formed between hRSV structural proteins, M and M2-1, and the components of the RNP complex, the genomic RNA, N, P, and L, during replication and assembly through correlative imaging strategies. Cryo-immuno-EM approaches and hRSV RNA-specific probes will be used to further define the organization of the structural proteins, M and M2-1, and the RNP complex during cryo-ET analysis of cryo-preserved virions and virus- infected cells.